CD8+ cells from HIV-1 infected individuals have been shown to suppress HIV-1 replication both through an MHC-restricted cytolytic pathway as well as through a non-cytolytic pathway. The latter pathway has been shown to be independent of HLA, does not require cell to cell contact and is mediated through soluble factors, hence the original name CD8+- cell antiviral factors (CAF). It now appears that immune cells, particularly CD8+ cells, make a number of factors that have inhibitory effects against HIV-1, several of them through interactions with an increasing number of chemokine co-receptors. These factors include the beta-chemokines RANTES, MIP-1alpha and MIP-1beta as well as macrophage derived chemokine (MDC). Herpesvirus saimiri (HVS)-transformed CD8+ cells have proven to provide a continuous source of supernatant with potent HIV-1 inhibitory activity. We have characterized this activity in HVS-transformed CD8+ cells from HIV-1 infected children with differing disease progression as well as CD8+ transformed cells from uninfected individuals. The inhibitory activity of unfractionated supernatant from these cell lines is potent and broad as well as distinct from the previous described factors. Supernatants inhibit T- tropic as well as M-tropic isolates; M-tropic inhibition in macrophages is consistently more effective then a cocktail of beta-chemokines at high concentrations. There is no correlation between level of inhibition and levels of any of these chemokines. Through sequential size fractionation, concentration, ion exchange and reverse phase chromatography we have purified and obtained partial amino acid sequence of at least one of these factors. Full inhibitory activity against HIV-1 Ba-L replication in macrophages is retained in the single 8kd protein at a concentration of approximately 30 mug/ml. This proposal will further characterize this unique and potent soluble factor by completing the cloning of the single 8kd protein isolated from a CD8+ HVS- transformed cell line, expression and purification of the inhibitory protein in a bacterial expression system, determination of the mechanism of inhibition by the recombinant and native protein and addressing issues related to the potential role of the protein in infection by examining major cell source in infected and uninfected individuals and through collaborations with the Laboratory of Genomic Diversity, NCI investigating the potential role in infection and progression. This unique cell culture system has provided a valuable source of continuous production and characterization of these natural inhibitors of HIV-1.